title: Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression.
abstract: Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.
authors.affiliation: University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, Minneapolis, Minnesota, United States of America.
authors.forename: Hai Dang
authors.initials: HD
authors.lastname: Nguyen
authors.forename: Jordan
authors.initials: J
authors.lastname: Becker
authors.forename: Yee Mon
authors.initials: YM
authors.lastname: Thu
authors.forename: Michael
authors.initials: M
authors.lastname: Costanzo
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Koch
authors.forename: Stephanie
authors.initials: S
authors.lastname: Smith
authors.forename: Kyungjae
authors.initials: K
authors.lastname: Myung
authors.forename: Chad L
authors.initials: CL
authors.lastname: Myers
authors.forename: Charles
authors.initials: C
authors.lastname: Boone
authors.forename: Anja-Katrin
authors.initials: AK
authors.lastname: Bielinsky
mesh_terms: D004247:DNA; D000072481:DNA Ligase ATP / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C000607890:CDC9 protein, S cerevisiae; D004268:DNA-Binding Proteins; C022262:Okazaki fragments; D018809:Proliferating Cell Nuclear Antigen; C400213:RAD59 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D000072481:DNA Ligase ATP